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Centro de Investigaciones sobre Desertificación (CSIC/ UV/GV), Camí de la Marjal s/n, 46470 Albal (Valencia), Spain
Custodia Cano
Concepción Azcón-Aguilar
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), calle Profesor Albareda 1, 18008 Granada, Spain
Julie Samson
Andrew P. Coughlan
Yves Piché
Centre de Recherche en Biologie Forestière (CRBF), Pavillon Charles-Eugène-Marchand, Université Laval, Québec, G1K 7P4 Canada
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ABSTRACT |
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A new in vitro experimental system was developed to study the morphogenesis of discrete regions of a single extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, growing simultaneously in six different agar-based media. The media were (i) unamended water agar (WA), (ii) WA+PO43– (PO43–), (iii) WA+NO3– (NO3–), (iv) WA+NH4+ (NH4+), (v) WA+NH4++MES (NH4++MES) and (vi) minimal medium (M, complete nutrients). Each medium was amended with the pH indicator bromocresol purple. The extraradical mycelium of the fungus showed between-treatment differences in morphogenesis, architecture, formation of branched absorbing structures (BAS) and sporulation. Extraradical hyphae that developed in WA or PO43– compartments exhibited an economic development pattern, in which runner hyphae radially extended the external colony. Extraradical hyphal growth in the NO3– compartments was characterized by increased formation of runner hyphae, BAS and spores and an alkalinization of the medium. In the two NH4+-amended media (NH4+, NH4++MES), sporulation was suppressed and considerable morphological changes were noted. These results show the plasticity of G. intraradices that lets it efficiently exploit an heterogeneous substrate.
Key words: branched absorbing structures (BAS), Glomus intraradices, monoxenic culture, nitrogen source, pH, sporulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) with soil-borne fungi in the phylum Glomeromycota (Schüssler et al 2001). The wide environmental tolerance of many taxa suggests that they have the capacity to adapt to different environmental conditions.
Arbuscular mycorrhizal fungi are obligate biotrophs, being unable to complete their life cycle in the absence of a host plant (Azcón-Aguilar et al 1998). After contact with a suitable host, inter- and/ or intracellular colonization of the root cortex occurs (Bonfante-Fasolo 1984, 1987, Smith and Smith 1997). Establishment of the symbiosis allows the completion of the AM fungal life cycle. This involves formation of an extraradical hyphal phase that colonizes the soil in the vicinity of the host root. These hyphae form characteristic structures including branched absorbing structures (BAS, formerly named arbuscule-like structures, ALS; Bago et al 1998b, c), spore-associated BAS (BAS-s; Bago et al 1998b, c) and spores. The extraradical mycelial network increases the nutrient uptake surface of the host plant and allows a more efficient extraction of phosphorus, nitrogen and certain micronutrients (Smith and Read 1997).
Several studies have highlighted the metabolic capacities of the AM extraradical phase (reviewed by Jakobsen 1995; see also Bago et al 1996, 2002, 2003, Olsson et al 2002). However, little is known about the effect of the heterogeneous soil environment on the extraradical mycelium (ERM) and its subsequent effects on nutrient uptake by AM fungi. Friese and Allen (1991) were the first to study extraradical hyphal architecture in rhizospheric soil. However recent studies by Bago et al (1998a, b, c) using AM monoxenic cultures (in vitro dual AM fungus and root-organ cultures) indicated that the AM extraradical mycelium is more complex than was reported by Friese and Allen (1991). Cui and Caldwell (1996a, b) showed that extraradical AM hyphae are equally efficient in transporting soil phosphate and nitrate, irrespective of the patchiness of the substrate. Therefore, we hypothesize that hyphal development and colony architecture of AM fungi vary on a microscale to maximize growth and nutrient uptake within heterogeneous substrates. Such responses have been shown to occur with other soil-borne fungi such as ectomycorrhizal (Bending and Read 1995) and saprophytic fungi (Bailey et al 2000, Fomina et al 2003, Ritz 1995). In particular, the work by Bailey et al (2000) indicates that fungal colony development in a given substrate depends mainly on nutrient distribution and availability but that it also is influenced by the intrinsic morphogenesis of the fungal colony, which includes the radial density of hyphal growth, aggregation into strands and the degree of branching and anastomosing. Our hypothesis also is supported by a recent study that shows the differential growing strategy of various AM fungi when colonizing and exploiting a given substrate (Smith et al 2000). The aim of the present work was to study morphological and developmental changes to the architecture of the extraradical mycelium of Glomus intraradices Smith and Schenk subjected to different nutritional conditions in a patchy environment.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The central (60 mm) plate was filled with 20 mL of sterilized (121 C, 20 min) minimal medium (Chabot et al 1992). The nutrient content of this medium (in mg/L of distilled water and molecular amounts) was: MgSO4·7H2O, 731 (29.6 mM); KNO3, 80 (7.9 mM); Ca(NO3)2·4H2O, 288 (12.2 mM) (total N content = 45.23 mg/L); KCl, 65 (8.71 mM); KH2PO4, 4.8 (1.37 mM); Na-FeEDTA, 8 (4.36mM); KI, 0.75 (4.51 mM); MnCl2·4H2O, 6 (30.3 mM); ZnSO4·7H2O, 2.65 (9.2 mM); H3BO3, 1.5 (24.3 mM); CuSO4·5H2O, 0.13 (0.52 mM); Na2MoO4·2H2O, 0.0024 (9.9 µM); glycine, 3 (3.99 mM); thiamine, 0.1 (29.7 µM); pyridoxine, 0.1 (48.6 µM); nicotinic acid, 0.5 (0.4 mM); myoinositol, 50 (27.7 mM); and sucrose, 10 g/L (29.2 mM). The central plate, referred to as the culture compartment (FIG. 1a, CC), was used to establish the mycorrhizal symbiosis. This consisted of a monoxenic culture (Bécard and Fortin 1988) of the AM fungus Glomus intraradices Schenck & Smith (DAOM 197198, Biosystematic Research Centre, Ottawa, Canada), grown with a carrot (Daucus carota L.) root-organ culture (clone DC-2). Monoxenic cultures were started by placing a 5 cm-long root in the culture compartment. A cube of medium from a previous monoxenic culture, containing approximately 100 spores, fragments of mycorrhizal roots and external hyphae, was placed near the root apex.
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, HC), were filled with 7 mL of one of six different sterilized (121 C, 20 min) culture media. These media consisted of 0.8% Bacto-Difco agar in distilled water either (i) unamended (water agar [WA]) or amended with (ii) 4.8 mg/L KH2PO4, (iii) 326 mg/L KNO3 (45.23 mg/L total nitrogen), (iv) 214 mg/L (NH4)2SO4 (45.23 mg/L total nitrogen), (v) 214 mg/L (NH4)2SO4 in a MES solution (10 mM, pH = 6) and (vi) nutrients to provide the same composition of the minimal medium. All six hyphal compartment media were amended (1%) with a solution (1% in distilled, sterile water; Bago et al 1996) of the pH indicator bromocresol purple to follow pH changes during the study (Bago et al 1996). After autoclaving, pH values of the different culture media were: minimal medium, 5.7; PO43–, 6.0; NO3–, 6.0; NH4+, 6.2; NH4++MES, 5.8; and WA, 6.2. Samples (n = 5) of the six media in which neither roots nor the fungus were cultured (referred to as color controls) were maintained under identical conditions as the cultures.
The space between the six hyphal compartments and the culture compartment was filled with bromocresol purple-free WA (pH 6.0) (FIG. 1a, Interm), so that hyphae that grew out of the culture compartment could develop in WA medium and then into the different test media. Ten replicates were set up and the plates incubated in the dark at 25 C for 84 d. Roots periodically were cut to prevent them developing in the surrounding WA media or HC.
Observation and measurements of fungal growth. –
At the end of the experiment, mean total runner hyphal length was calculated using a 2 x 2 mm grid (Marsh 1971) and the total numbers of BAS and spores were determined. All measurements were done under a Nikon AFX stereomicroscope. Microphotographs (Kodak 100 ISO film) of hyphal morphology and extraradical structures in the different culture media were taken with a Leica DMRB microscope fitted with a Leica MPS-60. Results were analyzed statistically using the Fisher’s Protected LSD test (P 
0.05).
pH measurements. –
Nondestructive pH measurements of the HCs were made according to Bago et al (1996). Absorbance of the culture media contained in each HC was measured (Shimadzu UV-visible spectrophotometer, UV 1603), without removing them from the Petri plates, at two wavelengths, 589 and 429 nm. The ratio between the two measured absorbances (i.e., A589/A429) was obtained, and the pH of the medium, y, was calculated using
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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After 28 d, extraradical hyphae extensively had colonized the 10 culture compartments. Approximately 14 d later, extraradical hyphae crossed over the plastic boundary of the culture compartments and developed in the external, bromocresol-free WA media (TABLE I). Extraradical hyphae also began to develop in the different HC. We observed an alkalinization (color change to purplish-violet) in the NO3– HC colonized by extraradical hyphae. A similar but weaker color change occurred in the minimal medium (which also contained NO3– as the sole nitrogen source). No detectable color (pH) changes occurred in compartments containing PO43– or WA after colonization by extraradical hyphae or in those containing the buffered NH4+ medium (NH4+ + MES). Unbuffered ammonium-amended compartments (NH4+) remained yellow (acidic) throughout the experiment.
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). Results showed that hyphae developing in compartments containing NO3– raised the pH of the medium by 0.5 (minimal medium) and 1.0 (NO3–) compared to their respective color control plates. The yellow observed in NH4+HC, where some extra-radical hyphae were developing, corresponded to a pH decrease of 0.4 when compared to the color control plates but of 1.3 pH units when compared to the initial pH of the medium (before the incubation period, see Methods). The pH of the rest of the HC did not differ by more than 0.1 from the pH of their respective color control plates, except for WA compartments, which showed a decrease of 0.3.
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). This contrasted with the reduced fungal development in the HC, particularly in the NH4+ treatments (see below).
When developing in WA (FIG. 1c, d), PO43– (FIG. 1e, f) or NO3– (FIG. 2e, f), the extraradical mycelium was highly organized. The mycelium consisted of thick (10–15 µm diam), leading runner (Friese and Allen 1991, Bago et al 1998b) hyphae that branched at approximately 45° (FIG. 1c, e) to produce thinner (1–7 µm diam), higher order, runner hyphae, resulting in a radial extension of the mycelium (FIG. 1e). Under this developmental pattern, runner hyphae showed apical dominance and constituted the framework of the colony. Branched absorbing structures (BAS) and spore-associated BAS (BAS-s; Bago et al 1998b, c) developed at relatively regular intervals on second and higher order runner hyphae (FIGS. 1d, f, 2f). Spores also were produced commonly by BAS-s (Bago et al 1998b) along runner hyphae (FIG. 2c–f).
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). Apical dominance was lost, and the mycelium frequently appeared tangled or interwoven (FIG. 2a, b, arrows; FIG. 2c). Leading runner hyphae were barely distinguishable (FIG. 2a, b) and BAS were less compact, merging within the profusely branched fungal colony. However, after 1–2 wk apical dominance recovered (FIG. 2d). Highly branched, leading runner hyphae re-appeared, and BAS formed at regular intervals (FIG. 2d, arrows).
Mycelial architecture was altered markedly when the fungus developed in the acidic NH4+ medium (FIG. 3a–d). Leading runner hyphae still branched at approximately 45° (FIG. 3a), but branches were thicker (approximately 10–12 µm diam versus 5–7 µm in other treatments, see above) and colony architecture appeared disrupted. Cytoplasmic protrusions occurred at different sites, especially at apices, which frequently appeared lysed (FIG. 3b, c, arrows). Runner hyphae occasionally formed short ramifications at irregular intervals (possibly atrophied BAS; FIG. 3b, white arrows). Coils of hyphae, which consisted of closely aligned, cytoplasm-filled hyphae, were observed in some of the replicates (FIG. 3c, d). In the MES-amended compartments, morphological changes also were evident but were different than those observed in NH4+ treatment (FIG. 3e, h); upon contact with the NH4+ + MES medium, the leading runner hyphae lost apical dominance (FIG. 3e, arrow) and branched so profusely that it was impossible to follow its growing pattern (FIG. 3h). Extraradical hyphae became much thinner and interwoven (FIG. 3h) and growth was restricted to the medium closest to the boundary of the HC, while the center of the compartment remained almost uncolonized.
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) were 1.5–2 times larger than in the other treatments and less compact than those that developed in WA (FIG. 1d), PO43– (FIG. 1f) and NO3– (FIG. 2f) compartments. In NH4+ + MES treatments, BAS were indistinguishable due to the profuse branching of the fungus (FIG. 3h; see TABLE II, footnote iv), and as outlined above in NH4+ compartments BAS were rare (TABLE II) and atrophied (FIG. 3b). The formation of BAS was stimulated most in NO3–, whereas WA, minimum medium and PO43–, treatments showed an intermediate degree of BAS formation (TABLE II).
Sporulation was increased significantly in the NO3– treatment (TABLE II, FIG. 2e). This effect also was observed in preliminary tests using similar experimental conditions but with monoxenic cultures of G. intraradices on untransformed tomato (Lycopersicon esculentum Mill.) root organs. Sporulation was lower in WA compartments, further reduced in minimal medium and PO43–, and suppressed in NH4+ compartments (either buffered or unbuffered, TABLE II). Although variable in all the media tested, spore size was particularly heterogeneous in minimal medium (FIG. 2c; 84% of them ranged from 15 to 40 µm, 14%, from 40 to 65 µm). However, spores looked viable and their lipid contents appeared similar irrespective of size. In all replicates, G. intraradices preferentially sporulated (1053 ± 330 spore/cm2) in the bromocresol-free WA medium surrounding the HC (FIGS. 1b, c, 3e). The morphological features described throughout were consistent in the 10 replicate plates.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Indeed, fungi exhibit marked phenotypic plasticity, quickly adapting to changing conditions, and their hyphae are particularly well suited to heterogeneous environments. In particular, arbuscular mycorrhizal (AM) fungi exhibit two distinct mycelial phases: an intraradical phase (intraradical mycelium, IRM), developing under relatively homogeneous conditions in the root cortex (Smith and Read 1997), and an extraradical phase (extraradical mycelium, ERM), developing under temporally and spatially heterogeneous conditions in the soil. Intraradical hyphae alter their morphology, physiology and probably their genetic expression to meet the constraints imposed by host root cells (Smith and Smith 1997). This prevents rejection of the association and allows establishment of a fully functional symbiosis. The extraradical mycelium of AM fungi also might be expected to adapt its growth pattern and metabolic capabilities to explore and exploit different soil microenvironments (Bago 2000).
The extent of spread of the extraradical AM hyphae depends on the fungal species and environmental conditions (van Bruggen et al 2000, Smith et al 2000) ranging from soil phosphorus concentration (Abbott et al 1984, Abbott and Robson 1985) to atmospheric CO2 (Klironomos et al 1998). Cui and Caldwell (1996a, b) demonstrated that the AM fungal ERM facilitates the acquisition of phosphate by plants growing in enriched soil patches. The results presented here demonstrate that different zones of the same AM ERM can develop differently to explore and exploit the surrounding substrate. These morphogenetic changes are localized (and sometimes transient) within the fungal colony and are influenced directly by the presence of a nutrient (or combination of nutrients). It is interesting to note that the growth of some hyphae into one hyphal compartment (HC) did not influence hyphal morphogenesis of the rest of the colony. The plasticity of the ERM might be an important strategy for adaptation and survival in a diverse range of ecosystems and within heterogeneous substrates. Although the experimental system used (monoxenic AM cultures) is somewhat artificial, our results, as well as those of other previously published studies that also used monoxenic cultures, seem to reflect accurately the morphogenetic processes known to occur in AM fungi when growing in soil.
When growing with little NO3–, PO43–) or no (WA) nutrients, extraradical AM hyphae exhibited a well organized and economic development pattern, probably designed to maximize exploration and exploitation of the medium and allow the production of spores outside the zone of influence of the colonized root. This pattern, similar to that described by Bago et al (1998a, b), is based on the formation of runner hyphae, which radially extend the fungal colony, and from which small, compact branched absorbing structures (BAS) develop at regular intervals. These latter structures have been proposed as preferential nutrient uptake structures (Bago et al 1998c). Thus, while runner hyphae rapidly extend the colony to find either zones with more nutrition within the substrate or a new host root to colonize, BAS immediately form to absorb nutrients and spores develop in older parts of the fungal colony that probably have switched from assimilative to reproductive metabolism. The spore contents are mainly carbonaceous compounds (stored as lipids and glycogen; Beilby and Kidby 1980, Jabaji-Hare 1988, Bonfante et al 1994), which are acquired by the intraradical hyphae from the host plant (Shachar-Hill et al 1995) and transported toward the extraradical mycelium (Pfeffer et al 1999, Bago et al 2002, 2003). However, spore formation also requires other nutrients, one of the most important being nitrogen, a principal component of chitin, which is abundant in the spore wall. The idea that nitrogen is taken up by the fungus (probably via BAS) from the culture medium during assimilative growth is supported by the enhanced BAS production observed on extraradical hyphae growing in NO3–-amended media. A further indication of NO3– uptake by the fungus in the HC comes from the observed alkalinization of the NO3–-containing media (NO3–, minimal medium), which also was observed by Bago et al (1996). The absence of pH changes in PO43– medium suggests that other mechanisms apart from symport/antiport were active on the fungal hyphae in this treatment (e.g., organic acid excretion), which could balance the otherwise expected alkalinization of the culture medium.
Fungal sporulation can be enhanced by increasing the C:N ratio of the culture media (Delatorre and Cardenascota 1996, Engelkes et al 1997, Pascual et al 1997, Yu et al 1998). After 84 d, the presence of an adequate supply of carbohydrates (exported from the intraradical mycelium) combined with a possible depletion of media nitrogen (through fungal NO3– up-take, Bago et al 1996) could explain the observed increase in spore production in the NO3– HC. Although NO3– is the sole nitrogen source in minimal medium HC, this culture medium also contains sucrose; such an exogenous carbon source, which the extraradical fungal structures are unable to acquire and metabolize (Pfeffer et al 1999, Bago et al 2002), seems to hinder spore production by G. intraradices ERM, as has been shown by St.-Arnaud et al (1996).
When a balanced mix of mineral nutrients is available in the medium (e.g., in minimal medium HC) uptake becomes the principal function of the mycelium, rather than exploration or reproduction. Intensive substrate exploitation probably was achieved by increased hyphal branching and a temporary loss of apical dominance by the runner hyphae in the minimal medium treatment. The fact that hyphae were in an assimilative rather than in a reproductive phase could explain the low spore number in this treatment. When growing in batch culture, fungal growth is unrestricted until nutrient depletion or alteration of other culture conditions inhibit it (Trinci et al 1994). It is interesting to note that after a certain time the morphogenesis of some hyphae in the minimal medium compartments changed to resemble those growing in low-nutrient media. This probably is explained by nutrient depletion of the minimal medium.
Although initially adjusted to 6.2, the pH of NH4+ HC fell sharply before fungal development within them. This decline probably was caused by the formation of carbonic acid (CO2 + H2O 
HCO3– + H+) in the NH4+-amended media after a build up of CO2 produced by the roots and hyphae in the culture compartment. Although production of this weak acid was not enough to reduce the pH in the other culture media, the fact that an (NH4)2SO4 solution in water trends to be acidic (as observed in the NH4+ color controls, which spontaneously drop almost 1 pH unit; TABLE II) seemed to be enough to shift the already weak chemical equilibrium of the medium.
Development of external hyphae of G. intraradices in NH4+ HC reflects the adaptive changes of this fungus when growing under adverse conditions. Changes in extraradical fungal morphology (e.g., infrequent and deformed BAS, formation of coiled hyphae and protruded apices and a suppression of sporulation) were observed when NH4+ was the only nitrogen form in the culture medium under acidic conditions. AM colonization has been reported in plants growing in mine spoils at pH 2.7 (Daft et al 1975, see also Heijne et al 1996). Using monoxenic cultures of G. intraradices Coughlan (1998) found that external hyphae stopped growing immediately on reaching HC containing WA medium adjusted to pH 4. Clark and Zeto (1996) reported a sharp reduction in intraradical arbuscules or vesicles in Glomus-colonized maize roots when plants were grown in acidic soil, results supported by Tiwari et al (2002) using AM monoxenic cultures. Finally, van Aarle et al (2002) have shown that low pH negatively influenced both extraradical hyphal spread and (possibly) spore formation in two AM fungi grown in soil, even though the roots of the host plants were not exposed to the same pH. These reports agree with the results obtained in our work. However, it remains unclear whether the morphological changes presented here were caused by low pH alone or whether the nitrogen source also played a role.
In an attempt to clarify this point, we prepared NH4+ HC containing 10 mM of the pH buffer MES. However, the results obtained for this treatment (i.e., loss of apical dominance and profuse runner hyphal growth) suggests that the changes observed were caused by the MES buffer rather than pH stabilization. Vilariño et al (1997) observed a slight increase in root colonization and a strong effect on ERM development when AM soil cultures were amended with MES. The authors attributed these results first to a possible effect of the buffer on soil micro-organisms. However, the monoxenic conditions used in the present study indicate that the enhanced growth observed in the MES-buffered medium must be due, at least in part, to other mechanisms. Vilariño et al (1997) also suggested that sulfur in the MES buffer (32 g per mol) might be responsible for the increased growth. This hypothesis, supported by previous studies performed in AM fungi grown axenically in the presence of sulfuric compounds (Hepper 1984, Bago 1990) seems more likely. In our study 2.6 mg sulfur per HC were added as MES. Therefore, it is possible that sulfur has an important effect on ERM development, but more research is needed to prove this.
Branched absorbing structures are formed by extraradical hyphae of AM fungi only after a successful symbiosis has been established with a host root (Bago et al 1998c). Our observations support the idea that BAS are intrinsic features of extraradical hyphae because they were formed under all experimental conditions tested. External conditions, however, strongly influenced their morphology. In contrast, spore formation seems to depend on nutrient (mainly nitrogen) availability and/or environmental conditions, with low pH and/or the presence of ammonium suppressing sporulation.
Finally, with regard to sporulation, why did spore production increase threefold in the bromocresol-free WA surrounding the HC, compared to the maximum sporulation rate obser ved within a HC (NO3–)? Although a negative effect of bromcresol purple on spore formation cannot be ruled out, it is probable that the extraradical hyphae of AM fungi differentiate into an exploitative (absorptive) mycelium in zones where nutrients are available. The nutrients absorbed then would be moved to older zones of the fungal colony where sporulation can be initiated. Such sporulation likely is a consequence of various nutritional and physiological conditions, such as the above-mentioned increased C:N ratio and/or colony aging.
In conclusion, our results highlight the ability of the ERM of Glomus intraradices to adapt its hyphal morphology and architecture in discrete microsites to efficiently exploit a given substrate. These results should be further tested by using different AM fungal isolates. New studies to clarify the discrete influence of nutrients, pH and environmental conditions on the differential morphogenesis of the AM extraradical mycelium also are needed.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address: Departamento de Microbiología del Suelo, Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), calle Profesor Albareda 1, 18008, Granada, Spain.
1 Corresponding author. E-mail: abago{at}eecs.csic.es
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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